Therapeutic agent for disc herniation

ABSTRACT

The present invention provides a therapeutic agent for disc herniation, which has extremely few adverse side effects, can achieve a prolonged pain-ameliorating effect when administered in only a single dose, and can exhibit a high therapeutic effect and high safety in clinical applications. The present invention relates to a therapeutic agent for disc herniation, which is characterized by containing chondroitinase ABC as an active ingredient and being administered in such a manner that the ingredient can be administered into a human disk in an amount of 1-8 units per disk.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation of U.S. application Ser. No.14/491,792, filed on Sep. 19, 2014, allowed, which is a Division of U.S.application Ser. No. 13/993,919, filed on Jun. 13, 2013, abandoned. U.S.application Ser. No. 13/993,919 is a 35 U.S.C. § 371 U.S. national entryof International Application PCT/JP2011/006938 (WO 2012/081227) havingan International filing date of Dec. 13, 2011, which claims under 35U.S.C. § 119(a) the benefit of Japanese Application No. 2010-277490,filed Dec. 13, 2010, the entire contents of which are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to a therapeutic agent for disc herniationcontaining chondroitinase ABC as an active ingredient.

BACKGROUND ART

Disc herniation is a disease that causes leg pain, low back pain, andthe like due to the pressure on nerves of spinal cords, and the like,attributed to protrusion of the disc tissue into the vertebral canalbecause the nucleus pulposus perforates the anulus fibrosus presenttherearound. It has been reported that the therapeutic principle is aconservative therapy and approximately 90% of cases have been cured bysuch a conservative therapy. As the conservative therapy, there havebeen performed various treatments, such as rest, bed rest, medication(non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, musclerelaxants), spinal orthosis (corset), traction therapy, thermotherapy,epidural block, nerve root block, and exercise therapy. If noamelioration is seen in these conservative therapies, a surgical therapyis selected, and surgical operations have been applied to 10-30% of allpatients with lumbar disc herniation. In recent years, chemonucleolysishas been designed in order to reduce the invasion and load due tosurgery.

Chemonucleolysis is a method of injecting an enzyme into the disc tobring about the nucleolysis of nucleus pulposus and reduce the internalpressure of the disc so that the pressure on the spinal nerve roots isreduced.

So far, a method of using chymopapain as an enzyme to be injected intothe disc has been reported, and its effectiveness has been reported in1964 (Non-Patent Document 1). However, chymopapain also acts on thesurrounding tissue of the disc including not only the nucleus pulposusbut also the spinal cord, and its sale as a medicine has beendiscontinued at present because severe neurological complications(paraplegia, transverse myelitis, cerebral hemorrhage, subarachnoidhemorrhage, quadriplegia, etc.) were found (Non-Patent Document 2).

Therefore, any medicines for chemonucleolysis are currently notcommercially available and the development of a medicine that is able tosafely perform chemonucleolysis has been desired.

It is considered that chondroitinase ABC decomposes theglucosaminoglycan chains of proteoglycans present in the nucleuspulposus (such as chondroitin sulfate chains and hyaluronic acid chains)to reduce the intradiscal pressure due to the reduction in high waterretention ability of proteoglycan, resulting in reduction of thepressure on the spinal nerve roots. In addition, unlike chymopapain, itwas reported in 1985 that chondroitinase ABC was expected as a safemedicine with almost no injury to such neural tissue surrounding thedisc and use of chondroitinase ABC as an enzyme to be injected into thedisc was tried (Patent Document 1, Non-Patent Document 3, Non-PatentDocument 4).

However, since the disc also has originally a role as a cushion tosupport the weight applied to the spine, the cushion function that thedisc should have originally had is impaired if the nucleus pulposus isremoved in excess. In fact, in patients with decreased disc height by atleast 30% by surgical treatment, it is shown that there is a possibilityof back pain that is persistent (Non-Patent Document 5). Therefore, ifthe nucleus pulposus undergoes nucleolysis excessively even inchemonucleolysis by administration of chondroitinase ABC, the cushionfunction of the disc that should have originally had is impaired toresult in leading to a possibility of adverse side effects.

In addition, since chondroitinase ABC is a heterologous protein that isnot present in humans, it is necessary to succeed in a therapy in only asingle dose rather than multiple doses, also from the viewpoint ofprevention of anaphylactic shock or the like. Completion of the therapyin a single dose administration means that it is required to show asignificant therapeutic effect in only a single dose administration andderive the optimal dose with few adverse side effects because multipledose administration cannot be performed until a complete recovery.

In addition, a lot of studies on disc degeneration have been carried outin animal models, but more attention and study effort were required toapply the results in animal models to the study of human diskdegeneration due to the fact that disks vary among different animalspecies (Non-Patent Document 6).

The special nature of a single dose administration and the difficulty inapplying animal models to humans as described above makechemonucleolysis with chondroitinase ABC difficult, and, in fact,practical use of chondroitinase ABC has not yet been realized even todaythough 25 years have passed since 1985 described above.

Patent document 1 describes that disc herniation is treated byadministering chondroitinase ABC into a disc so that the nucleuspulposus undergoes the nucleolysis and a therapeutic effect on discherniation was obtained by administering chondroitinase ABC in an amountof 100 units per disk into a human disk. However, its effective dose hasnot yet been determined because the effect is not sufficient and theinvestigation on the adverse side effects has not been made. Inaddition, Non-Patent Document 3 describes that a therapeutic effect ondisc herniation was obtained by administering chondroitinase ABC in anamount of 0.5 units per disk into a human disk. Non-Patent Document 4describes that chondroitinase ABC was administered into a dog disc in anamount of 0.5-1, 2.5-5, and 5-10 units per disk, but when this dose isconverted into the dose for humans, it becomes to be about at least 35units because the volume of human nucleus pulposus is approximately 70times compared to that of dog nucleus pulposus. However, a detailedstudy of adverse side effects after administration of chondroitinase ABChas not been carried out.

Therefore, the optimal dose of chondroitinase ABC to show the maximumtherapeutic effect and the minimum side effect has neither beendisclosed nor suggested.

PRIOR ART DOCUMENT Patent Document

Patent Document 1: U.S. Pat. No. 4696816

Non-Patent Documents

Non-Patent Document 1: Smith L. Enzyme dissolution of the nucleuspulposus in humans. JAMA 1964(2); 187: 137-40.

Non-Patent Document 2: US Federal Register. Monday Jan. 27, 2003;68(17):

3886-7.

Non-Patent Document 3: Rinsho Seikei Geka (The Journal of the JapaneseClinical Orthopaedic Association) Vol. 42, No. 3, pages 223-228, March,2007

Non-Patent Document 4: SPINE, 1991; 16(7): 816-19

Non-Patent Document 5: SPINE, 1996; 21(13): 1556-64

Non-Patent Document 6: Eur Spine J, 2008; 17: 2-19

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

As described above, it has been reported that chondroitinase ABC isuseful as an active ingredient of a medicine for chemonucleolysis, butchondroitinase ABC has not yet been put to practical use because seriousadverse side effects caused by the excess dissolution of the nucleuspulposus is also concerned and it is unknown whether or not a reliabletherapeutic effect can be exhibited in only a single dose and whether ornot there is a dose with no adverse side effects.

The problem of the present invention is to provide a therapeutic agentfor disc herniation, which has extremely few adverse side effects, canachieve a prolonged pain-ameliorating effect when administered in only asingle dose, and can exhibit a high therapeutic effect and high safetyin clinical applications.

Means for Solving the Problem

As a result of further intensive studies about the treatment withchondroitinase ABC by the present inventors, it has been surprisinglyfound that by the administration of chondroitinase ABC in a specificamount, adverse side effects can be reduced and a prolongedpain-ameliorating effect/a high therapeutic effect in clinicalapplications are exhibited, and thus the present invention has beencompleted.

That is, it has been found that not only therapeutic effects can beexpected but also adverse side effects can be reduced at a dose of 1-8units of chondroitinase ABC per disk into a human disk, and that anexcellent therapeutic agent for disc herniation that uses chondroitinaseABC and is practical can be provided.

The present invention relates to a therapeutic agent for discherniation, containing chondroitinase ABC as an active ingredient andbeing administered into a human disk in an amount of 1-8 units per disk.

Also, the present invention relates to a formulation for the treatmentof disc herniation, which formulation is used for the administration ofchondroitinase ABC into a human disk in an amount of 1-8 units per disk.

In addition, the present invention relates to a method for treating discherniation, comprising administering a formulation containing, as aneffective dose, 1-8 units of chondroitinase ABC to a patient with discherniation.

More particularly, the present invention is as follows.

(1) A therapeutic agent for disc herniation, which is characterized bycontaining chondroitinase ABC as an active ingredient and beingadministered in such a manner that the ingredient can be administeredinto a human disk in an amount of 1-8 units per disk.

(2) The therapeutic agent according to the above (1), wherein the discherniation is a lumbar disc herniation.

(3) The therapeutic agent according to the above (1) or (2), wherein thechondroitinase ABC is derived from Proteus vulgaris.

(4) A formulation containing chondroitinase ABC for treating discherniation by the administration into a human disk in an amount of 1-8units per disk.

(5) The formulation according to the above (4), wherein the formulationis a single dose formulation.

(6) The formulation according to the above (4) or (5), wherein theformulation is an injection.

(7) The formulation according to any one of the above (4) to (6),wherein the disc herniation is a lumbar disc herniation.

(8) A method for treating disc herniation, comprising administeringchondroitinase ABC to a human disk of a patient with disc herniation inan effective amount of 1-8 units per disk.

(9) Chondroitinase ABC for use as a therapeutic agent for discherniation, which is characterized by being used in such a manner thatit can be administered into a human disk in an amount of 1-8 units perdisk.

Effects of the Invention

In accordance with the present invention, there can be provided atherapeutic agent for disc herniation, which has fewer adverse sideeffects, is able to complete the therapy of disc herniation in only asingle dose, is safe, has a high therapeutic effect and a high clinicalusefulness, and is highly practical.

The present invention is intended to clarify for the first time theexistence of a dose that is safe and has a high therapeutic effect aswell as a clinical usefulness in chemonucleolysis with chondroitinaseABC and also clarify for the first time the limited range of the doseincluding 1-8 units, preferably 1-5 units, contributing much to apractical use of chemonucleolysis by the administration ofchondroitinase ABC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the transition of leg pain at the worst time(VAS) after administration of a therapeutic agent.

FIG. 2 is a graph showing the incidence rate of adverse side effects peradministration group of each unit.

FIG. 3 is a graph showing the average reduction rate for the disc heightper administration group of each unit.

MODES FOR CARRYING OUT THE INVENTION

The following describes embodiments of the present invention.

(1) Active Ingredient of the Therapeutic Agent of the Present Invention

Chondroitinase ABC used as an active ingredient for the therapeuticagent of the present invention is not particularly limited as long as itis an enzyme having an action of chondroitinase ABC. Although its originis also not particularly limited, chondroitinase ABC is preferablyderived from microorganisms and preferably derived from Proteusvulgaris.

The method for manufacturing such chondroitinase ABC is also notparticularly limited, and chondroitinase ABC may be manufactured, forexample, by culturing a microorganism such as Proteus vulgaris and thelike or by genetic engineering techniques using a DNA encoding thechondroitinase ABC.

Chondroitinase ABC as such is preferably an enzyme which is purified tothe extent that can be used as a medicine and does not contain anymedically unacceptable contaminants.

More specifically, such chondroitinase ABC has an enzymatic activity of270 units/mg protein or more and contains endotoxin, nucleic acid, andprotease in amounts not more than the respective detection limits. Suchchondroitinase ABC can be obtained by, for example, the method asdescribed in JP 6-153947-A.

Further, in the present invention, “1 unit” of chondroitinase ABC isreferred to as the amount of enzyme liberating 1 micromole (M) ofunsaturated disaccharide per minute when the enzyme was allowed to reactwith chondroitin sulfate C as a substrate at pH 8.0 and 37° C.

When chondroitinase ABC having an enzymatic activity of 270 units/mgprotein or more is used, proteoglycan at the targeted site can beappropriately degraded without affecting tissues around the targetedsite by administering it to a living body as an injectablepharmaceutical preparation. Thus, the enzyme can be a safe and effectivemedicine.

(2) Target Disease

Target disease of the therapeutic agent of the present invention is notlimited as long as it is disc herniation, and such a target disease ispreferably a lumbar disc herniation, and especially preferably, amongthese, a lumbar disc herniation occurring in the disc between the fourthlumbar vertebra and the fifth lumbar vertebra, or in the disc betweenthe fifth lumbar vertebra and the first sacrum.

(3) Administration Site/Method/Frequency

The therapeutic agent of the present invention is used to inject intothe nucleus pulposus present within the disc where disc herniationoccurs. The frequency of the injection is once.

Chondroitin sulfate chains and hyaluronic acid chains in the nucleuspulposus of the disc are degraded by this injection so that the highwater retention ability of proteoglycans is attenuated to result inreduction of the intradiscal pressure, leading to reduce the pressure onspinal nerve roots due to disc herniation, thereby to ameliorate discherniation.

(4) Dose

Chondroitinase ABC that is the active ingredient of the therapeuticagent of the present invention is administered at a dose of 1-8 unitsper disk. Of these, 1-6 units are preferably administered, 1-5 units aremore preferably administered, 1-3 units are more preferablyadministered, 1-2.5 units or 1.25-3 units are further preferablyadministered, 1.25-2.5 units are especially preferably administered, and1.25 units or 2.5 units are most preferably administered.

In particular, at a dose of 1.25-5 units, the therapeutic effect isalmost the same (see FIG. 1), and the incidence rate (%) of adverse sideeffects is minimal at a dose of 2.5 units (see FIG. 2). Thus, 1-5 units,preferably 1-3 units are required in order to ensure the effectivetherapeutic effect and safety in only a single dose.

(5) Dosage Form, etc.

The therapeutic agent of the present invention can be provided in adosage form that is usually employed as an injection. For example, thedosage form may be any of a solution form, a frozen form, and afreeze-dried form. An injection can be prepared by filling this into anappropriate container such as ampoules, vials, syringes for injections,etc. and sealing the container.

Further, when filling or sealing the therapeutic agent of the presentinvention in a suitable container, such as ampoules, vials, and syringesfor injection, in order to prevent a chemical reaction of thetherapeutic agent of the present invention, especially oxidation, thecontainer may be filled or sealed together with an inert gas such asnitrogen gas and rare gas.

The material of the container such as ampoules, vials, and syringes forinjections, which is able to fill and seal the therapeutic agent of thepresent invention, is not particularly limited as long as it does notaffect the therapeutic agent of the present invention and is apharmaceutically acceptable material.

It is possible to use known methods for formulation of the therapeuticagent of the present invention. In the formulation, otherpharmaceutically active ingredients and ingredients usually used inmedicines, such as conventional excipients, stabilizers, binders,emulsifiers, osmotic agents, buffers, pH regulators, isotonic agents,preservatives, soothing agents, colorants, or the like may be used aslong as they do not adversely affect the activity of chondroitinase ABCitself and do not adversely affect the action of chondroitinase ABC.

EXAMPLES

Hereinafter, the present invention will be described in more detail byway of Examples, but these are illustrative of the present invention andthe scope of the present invention is not intended to be limitedthereto.

Example 1 Production of Therapeutic Agent of the Present Invention (1)Production of Chondroitinase ABC

Chondroitinase ABC was produced by culturing Proteus vulgaris andpurifying the culture supernatant according to the method described inJP 6-153947-A.

The respective enzyme activities of chondroitinase ABC produced were allin the range of 270-480 units/mg protein. In addition, the contents ofendotoxin, nucleic acid, and protease in chondroitinase ABC were allequal to or below the detection limit.

(2) Formulation

The following three kinds of freeze-dried injectable formulations (A) to(C) containing the chondroitinase ABC having the following each unit andthe following formulation ingredients.

(A) The chondroitinase ABC of 5 units

(B) The chondroitinase ABC of 10 units

(C) The chondroitinase ABC of 20 units

Formulation ingredients:

Sodium hydrogen phosphate hydrate 1.125 mg Sodium dihydrogen phosphate0.3 mg Sucrose 5 mg Polyethylene glycol 3350 10 mg

(Note: Polyethylene glycol 3350 corresponds to macrogol 4000 listed inthe Japanese Pharmacopoeia.)

Further, a freeze-dried injectable formulation containing only theformulation ingredients (not containing chondroitinase ABC) prepared inthe same manner was used as a placebo.

(3) Dissolution before Administration

A ready-to-use solution (4 mL) having the following composition wasadded to each formulation of (A) to (C) (including a placebo) describedabove before use, so that the formulations were dissolved to prepare aformulation of 1.25 units/mL for administration, a formulation of 2.5units/mL for administration, and a formulation of 5 units/mL foradministration, respectively. A placebo formulation was prepared in thesame manner. Ready-to-use solution:

Sodium hydrogen phosphate hydrate 3.375 mg Sodium dihydrogen phosphate0.9 mg Sucrose 15 mg Polyethylene glycol 3350 30 mg (corresponding tomacrogol 4000 listed in the Japanese Pharmacopoeia) Sodium chloride 36mg Water for injection 4 ml

Example 2 Study on Patients with Disc Herniation 1. Subjects

Japanese patients (194 cases in total) with the age of 20-70 years andwith the following lumbar disc herniation were served as the subjects:

patients with protrusion herniation or subligamentous extrusionherniation (not protruding into the posterior longitudinal ligament), inwhom a lumbar disc herniation occurring in the disc between the fourthlumbar vertebra and the fifth lumbar vertebra or in the disc between thefifth lumbar vertebra and the first sacrum was confirmed by MRI andclinical symptoms were consistent with the location of the damaged nerveroots; and

patients in the case where a sixth lumbar vertebra is observed and inwhom the fifth lumbar nerve root or the first sacral nerve root isdamaged and clinical symptoms are consistent with the location of thedamaged nerve roots.

The above subjects were divided into each group of a placeboadministration group (47 subjects), an administration group of 1.25units of chondroitinase ABC (49 subjects), an administration group of2.5 units of chondroitinase ABC (49 subjects) and an administrationgroup of 5 units of chondroitinase ABC (49 subjects), and correspondingformulations as described above were administered respectively.

2. Method for the Administration of the Therapeutic Agent of the PresentInvention

Of each formulation (4 ml) for the administration as described inExample 1, 1 ml of such a formulation was used for the administration.Therefore, the amount of the enzyme which is to be administered is 1.25units/mL in the case of the formulation for the administration of 1.25units, 2.5 units/mL in the case of the formulation for theadministration of 2.5 units, and 5 units/mL in the case of theformulation for the administration of 5 units.

Using these formulations, a single dose of 1 ml each was injected intothe nucleus pulposus of the disc as shown below.

3. Evaluation (1) Pharmacological Evaluation (a) Pain Evaluation by theSubject (Visual Analog Scale: VAS)

At each time point of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 13 weeks, 26 weeks, 39 weeks and 52 weeks after administration ofthe formulation, a measurement about “the worst leg pain during the past24-hour period (VAS evaluation)” that was evaluated by the subjectoneself was performed (a measurement of the VAS value).

The VAS evaluation by the subject was performed before bedtime. On a 100mm straight line of “a pain evaluation sheet” in which “no pain” isdescribed on the left end of the straight line and “biggest pain felt inthe past” is described on the right end, the degree of pain was markedwith a point by the subject oneself.

The distance (mm) of from the left end of the same straight line to thepoint where the subject had marked was measured to evaluate the degreeof pain. The results of this evaluation were analyzed by a Dunnett typemultiple comparison test using a placebo group as a control.

In addition, the rate of change in pain (rate of change in VAS) wasevaluated. The rate of change in VAS was determined by obtaining a VASvariation by subtracting the VAS value at 13 weeks after administrationof the formulation from the VAS value before administration of theformulation, and dividing the VAS variation by the VAS value beforeadministration of the formulation. A covariance analysis (p<0.05) wasperformed for this VAS variation, using a placebo group as a control.

(b) Neurological Examination

A straight leg raising test (SLR test) was performed at week 13 afteradministration of the formulation, in order to examine the nervestimulation caused by disc herniation.

The SLR test is one of the neurological examinations of lumbar discherniation, and when the lower limbs were stretched, the case where thestraight leg raising angle is 70 degrees or less is determined to be“positive” and the case where the straight leg raising angle exceeds 70degrees is determined to be “negative”. Before administration of theformulation in this study, all subjects were positive.

The frequency of the positive and negative cases in the SLR test foreach group was summed up, and the ratio of negative cases in the SLRtest was examined by a Steel-type multiple comparison using a placebogroup as a control.

In addition, an evaluation of drug efficacy was carried out in 193 casesof the target subjects.

(2) Safety Evaluation

A safety evaluation was carried out in 194 cases of the target subjects.

The number of appearance and the incidence rate of adverse side effectswere determined. The evaluation was carried out until 13 weeks afteradministration of the formulation. However, among the adverse sideeffects, the following items of (a) and (b) were evaluated until 52weeks after the administration;

when compared to the value before administration of the formulation, (a)the cases in which the reduction rate of the disc height was equivalentto or more than 30% after such administration, and (b) the cases inwhich the vertebral posterior angle of the vertebral body was equivalentto or more than 5 degrees.

In addition, the safety evaluation of the item (b), such that thevertebral posterior angle of the vertebral body should be equivalent toor more than 5 degrees, is an index related to the instability of thedisc, which is established by The U.S. Food and Drug Administration(FDA).

The average reduction rate of the disc height in an administration groupreceiving each unit was determined 13 weeks after the administration ofthe formulation.

4. Result (1) Pharmacological Evaluation (a) VAS

FIG. 1 shows transition of the worst leg pain (VAS) after administrationof the formulation. The horizontal axis of FIG. 1 shows the time (weeks)after the administration, and the vertical axis shows the value (mm) ofVAS. The rhomboid mark (♦) in the graph shows the results of the placebogroup, the black circle mark (●) shows the results of an administrationgroup of 1.25 units of chondroitinase ABC, the square mark (▪) shows theresults of an administration group of 2.5 units of chondroitinase ABC,and the black triangle mark (▴) shows the results of an administrationgroup of 5 units of chondroitinase ABC.

A pain-inhibitory effect was observed from the first week in any of theadministration groups of the formulations. In particular, a significantpain-inhibitory effect (p<0.05) was observed from the first week in theadministration group of the formulation of 1.25 units of chondroitinaseABC, and the administration group of the formulation of 5 units ofchondroitinase ABC, compared to the placebo group. In addition, at weeks39 and 52 after administration, a significant pain-inhibitory effect(p<0.01 or p<0.001) was shown in all the administration groups, comparedto the placebo group. The pain-inhibitory effects are substantiallyequal in the administration group of 1.25 units, the administrationgroup of 2.5 units, and the administration group of 5 units, and it wasshown that such inhibitory effect was effective over a one year (52weeks). As a result, it was demonstrated that a single administration ofthe formulation showed a significant pain-inhibitory effect.

In addition, the rate of change in VAS at week 13 after theadministration was 66% in the administration group of 1.25 units ofchondroitinase ABC, 61% in the administration group of 2.5 units ofchondroitinase ABC, and 69% in the administration group of 5 units ofchondroitinase ABC, and a significant reduction in the rate of change inVAS was confirmed in any of the administration groups of the aboveunits, compared to that (45%) of the placebo administration group.

(a) Neurological Examination (SLR Test)

The negative rate in the placebo group was about 50%, but the negativerates in the administration groups were all 60% or more. In particular,the negative rate in the administration group of 1.25 units ofchondroitinase ABC has reached 80% or more.

In any administration groups, there was an increase in the proportion ofnegative response. Particularly, the proportion of negative response wassignificantly increased (p<0.01) in the administration group of 1.25units of chondroitinase ABC, compared to the placebo administrationgroup.

(2) Safety Evaluation

The incidence rate of adverse side effects is shown in FIG. 2. Thehorizontal axis in FIG. 2 shows each administration group, and thevertical axis shows the incidence rate (%) of adverse side effects. Theincidence rate of adverse side effects in the administration group of 5units of chondroitinase ABC was 61.2%, 44.9% in the administration groupof 2.5 units of chondroitinase ABC, and 46.9% in the administrationgroup of 1.25 units of chondroitinase ABC. From this, it was shown thatthe minimum value of the incidence rate of adverse side effects was inthe vicinity of 2.5 units per disk for the administration group and thepresence of a dose being able to cause minimal adverse side effects wasclarified.

Then, adverse side effects as the instability of the disc were studiedon (a) the cases where the vertebral posterior angle of the vertebralbody was equivalent to or more than 5 degrees and (b) the cases wherethe average reduction rate of the disc height was equal to or more than30%. The results are shown in Table 1 below.

TABLE 1 Placebo 1.25U 2.5U 5U 10U Administration group group group groupgroup group N = 47 N = 49 N = 49 N = 49 N = 6 Vertebral posterior 0 (0%)0 (0%) 0 (0%) 1 (2.0%) 2 (33.3%) angle of vertebral body Reduction indisc 0 (0%) 4 (8.2%) 4 (8.2%) 7 (14.3%) — height — : No data

As a result, 1 case (2% incidence rate) where the vertebral posteriorangle of the vertebral body was 5 degrees or more was observed in theadministration group of 5 units of chondroitinase ABC. On the otherhand, there was no case where the vertebral posterior angle of thevertebral body was 5 degrees or more in the administration group of 1.25units of chondroitinase ABC and in the administration group of 2.5 unitsof chondroitinase ABC. Further, in the administration group of 10 unitsof chondroitinase ABC as a comparative example, 2 cases (33.3% incidencerate) where the vertebral posterior angle of the vertebral body was 5degrees or more were observed, indicating that adverse side effectsrelated to the instability of the disc occurred in a high percentage,which was of no practical use.

There were 7 cases (14.3% incidence rate), where the reduction rate inthe disc height was 30% or more, in the administration group of 5 unitsof chondroitinase ABC, 4 cases (8.2% incidence rate) in theadministration group of 1.25 units of chondroitinase ABC, and 4 cases(8.2% incidence rate) in the administration group of 2.5 units ofchondroitinase ABC. The occurrence of the adverse side effects was foundto be decreased rapidly.

Next, the average reduction rate of the disc height in theadministration groups of each unit is shown in FIG. 3. The horizontalaxis in FIG. 3 shows each administration group, and the vertical axisshows the reduction rate (%) of the disc height.

As a result, it was found that the average reduction rates of the discheight in the administration groups of 5 units, 2.5 units, and 1.25units of chondroitinase ABC were all less than 30%. These administrationgroups each showed a great reduction rate of the disc height compared tothe placebo group, and it was shown that a therapeutic effect wasobserved. In addition, in the administration group of 10 units ofchondroitinase ABC in Comparative Example, the reduction rate was foundto be 30% or more.

In addition, an increase in IgG antibody titer against chondroitinaseABC, which is a side effect, was observed in 1 case in theadministration group of 2.5 units and in 1 case in the administrationgroup of 5 units. This increase in the antibody titer was not observedin the administration group of 1.25 units.

(Comparative Example) Safety Evaluation in Administration of 10 Units

Chondroitinase ABC of 10 units per disk was administered to patients (6persons) with lumbar disc herniation in the same way as above. As asafety evaluation, (a) the reduction rate in the disc height at week 12after the administration and (b) the case with the number and incidencerate of vertebral posterior angle of the vertebral body being equivalentto or more than 5 degrees were determined.

The reduction rate in the disc height at week 12 after theadministration is shown in FIG. 3. As a result, the average reductionrate in the disc height was 45.4% which was significantly greater thanthe value (30%) that might produce a problem in safety.

In addition, the results of the vertebral posterior angle of thevertebral body being equivalent to or more than 5 degrees were alsoshown in Table 1. As shown in Table 1, 33.3% of the patients who showedthe vertebral posterior angle of the vertebral body being equivalent toor more than 5 degrees were observed in the administration group of 10units of chondroitinase ABC. From these results, it is considered thatthe administration group of 10 units has a high risk related to theinstability of the spine.

The volume of the nucleus pulposus in humans and dogs was measured byMRI in order to estimate a dose of chondroitinase ABC effective tohumans from the dose of chondroitinase ABC in dogs as described in theNon-Patent Document 4.

(Reference Example) Comparison of Volume of Nucleus Pulposus Using MRI

T2 weighted images were obtained by MRI imaging of the disc between thefourth lumbar vertebra and the fifth lumbar vertebra in 9 healthyvolunteers (6 males, 3 females) and resected specimens of dogs (6animals), and the minor axis, major axis, and area of coronal image ofthe nucleus pulposus, and the minor axis of sagittal image of thenucleus pulposus were measured. The volume of the nucleus pulposus wascalculated (coronal cross-sectional area x sagittal cross- sectionalminor axis). The results are shown in Table 2 below.

TABLE 2 Volume of nucleus pulposus Ratio of average (mm³) (Calculatedvalue volume of nucleus Animal species from MRI image) pulposus in MRIimage Human 7852.2 ± 2041.2 Human/dog = 70.2 Dog (Beagle) 111.8 ± 70.3 Value = Average ± Standard deviation

5. Summary

The incidence rate of showing the vertebral posterior angle of thevertebral body being equivalent to or more than 5 degrees was 33.3% when10 units of chondroitinase ABC were administered, whereas it was foundthat such an incidence rate could be significantly reduced to 2% when ahalf dose, i.e. 5 units were administered.

In addition, while the average reduction rate of the disc height alsoreached even 45.4% when 10 units of chondroitinase ABC wereadministered, the reduction rate of the disc height was 17.6% in theadministration group of 5 units of chondroitinase ABC. Thus, it wasshown that the reduction in the disc height could be remarkablysuppressed by decreasing the dose from 10 units to the half dose, i.e. 5units.

The Non-Patent Document 3 describes that when 0.5 unit of chondroitinaseABC per disk was administered into the human disk, a rapid ameliorationin leg pain did not occur, indicating that 0.5 unit was a less dose, butdetailed studies on the adverse side effects were not performed and theclinically effective dose was unknown.

By administering chondroitinase ABC in a range of 1-8 units per disk,preferably 1-5 units per disk in accordance with the present invention,it is possible to reduce the adverse side effects while exhibiting asignificant amelioration in pain in only a single dose. Further, it wasfound that by selecting an administration of chondroitinase ABC in therange of 1-3 units, the adverse side effects could be further reducedwhile exhibiting a similar effect in ameliorating pain to a higher doseof 5 units.

On the other hand, Non-Patent Document 4 describes that even when 0.5-1unit of chondroitinase ABC per disk was administered into a dog disc,rapid reduction of the nucleus pulposus did not occur, indicating thatthe dose was too small. In addition, Non-Patent Document 6 describesthat the setting of scaling is necessary in the analysis of theexperimental results in animal models because the behavior of the discdepends on the size of the disc. Then, from the above reference example,it was confirmed that the volume of human nucleus pulposus was 70 timesas large as that of dog nucleus pulposus. In view of the descriptions ofthe Non-Patent Documents 4 and 6 and given that the volume of humannucleus pulposus was much larger than the volume of dog nucleuspulposus, even when 35-70 units, which were 70 times of 0.5-1 unit, ofchondroitinase ABC were administered, such a dose was estimated to benot enough.

In this way, the dose for humans estimated from the experimental resultsin dogs as described in the Non-Patent Document 4 is quite differentfrom the dose that was found in the present invention, and it wasextremely difficult to predict an effective dose for humans.

Thus, it was found that adverse side effects could be reduced by theadministration of 1-8 units of chondroitinase ABC per disk into a humandisk and disc herniation could be treated in only a single dose.

INDUSTRIAL APPLICABILITY

The present invention can provide a therapeutic agent for discherniation, which has extremely few adverse side effects, can achieve aprolonged pain-ameliorating effect when administered in only a singledose, and can exhibit a high therapeutic effect and high safety inclinical applications.

1-4. (canceled)
 5. A method for treating lumbar disc herniation,comprising: injecting a formulation consisting essentially ofchondroitinase ABC in an effective amount of 1-3 units per disc in asingle dose into a disc of a human patient with disc herniation.
 6. Themethod according to claim 5, wherein the disc of a human patient is adisc where a nucleus pulposus is present and where the disc herniationoccurs.
 7. The method according to claim 6, wherein the formulationcontains at least one ingredient selected from the group consisting ofan excipient, a stabilizer, a binder, an emulsifier, an osmotic agent, abuffer, a pH regulator, an isotonic agent, a preservative, a soothingagent and a colorant.
 8. The method according to claim 7, wherein theformulation is lyophilized.
 9. The method according to claim 5, whereinthe effective amount of chondroitinase ABC is 1.25 units per disc. 10.The method according to claim 9, wherein the disc of a human patient isa disc where a nucleus pulposus is present and where the disc herniationoccurs.
 11. The method according to claim 10, wherein the formulationcontains at least one ingredient selected from the group consisting ofan excipient, a stabilizer, binder, an emulsifier, an osmotic agent, abuffer, a pH regulator, an isotonic agent, a preservative, a soothingagent and a colorant
 12. The method according to claim 11, wherein theformulation is lyophilized.
 13. The method according to claim 12,wherein the chondroitinase ABC is derived from Proteus vulgaris.
 14. Themethod according to claim 12, wherein the formulation contains sucroseand polyethylene glycol
 3350. 15. The method according to claim 13,wherein the formulation contains sucrose and polyethylene glycol 3350.16. A method for treating lumbar disc herniation, comprising: injectinga formulation that comprises chondroitinase ABC in an effective amountof 1-3 units per disc in a single dose into a disc of a human patientwith disc herniation, wherein chondroitinase ABC is a sole activeingredient for therapeutic effect in lumbar disc herniation of theformulation.
 17. The method according to claim 16, wherein the disc of ahuman patient is a disc where a nucleus pulposus is present and wherethe disc herniation occurs.
 18. The method according to claim 17,wherein the formulation contains at least one ingredient selected fromthe group consisting of an excipient, a stabilizer, a binder, anemulsifier, an osmotic agent, a buffer, a pH regulator, an isotonicagent, a preservative, a soothing agent and a colorant.
 19. The methodaccording to claim 18, wherein the formulation is lyophilized.
 20. Themethod according to claim 16, wherein the effective amount ofchondroitinase ABC is 1.25 units per disc.
 21. The method according toclaim 20, wherein the disc of a human patient is a disc where a nucleuspulposus is present and where the disc herniation occurs.
 22. The methodaccording to claim 21, wherein the formulation contains at least oneingredient selected from the group consisting of an excipient, astabilizer, a binder, an emulsifier, an osmotic agent, a buffer, a pHregulator, an isotonic agent, a preservative, a soothing agent and acolorant.
 23. The method according to claim 22, wherein the formulationis lyophilized.
 24. The method according to claim 23, wherein thechondroitinase ABC is derived from Proteus vulgaris.
 25. The methodaccording to claim 23, wherein the formulation contains sucrose andpolyethylene glycol
 3350. 26. The method according to claim 24, whereinthe formulation contains sucrose and polyethylene glycol 3350.